The science industry, and the bioscience and environmental industries in particular, rely on the analysis of large numbers of samples for various studies. The need for rapid turnaround time coupled with the high costs of labor and chemical waste disposal have resulted in the development of automated array-based techniques that analyze samples using optical spectroscopy. Although these conventional absorption-based techniques are applicable to a wide spectrum of analytes, they have low sensitivity.
The two major types of optical spectroscopy currently used in array-based analysis include absorption spectroscopy and fluorescence spectroscopy. The most common technique is conventional absorption spectroscopy. Light at a given wavelength is transmitted through the sample, and the decrease in intensity relative to the original beam is monitored. The concentration of absorbing substance is determined using the Beer-Lambert law, which requires knowledge of the intrinsic absorptivity of the substance, the path length of light through the sample, and the ratio of incident and transmitted light intensities. Because direct measurement of absorption involves sensing a small decrease in the strength of a high background signal (i.e., the intensity of the unblocked light beam), conventional absorption spectroscopy is a low signal-to-noise technique. As a consequence, it has limited sensitivity (typically on the order of 10−3 absorbance units, corresponding to absorption of about 0.2% of the incident light by the sample). This analysis method does, however, have wide applicability. A large number of analytes absorb light with sufficient efficiency to be detected by a decrease in transmissivity.
The other major type of spectroscopy in common use with sample arrays is fluorescence spectroscopy. This technique also relies on absorption of incident light by the sample, but detection is based on the emission of light of lower energy (longer wavelength) as the absorber decays from the excited state. The background signal of the detector, therefore, is zero (except for “dark current” noise in the electronic circuitry), and the signal-to-noise is very high.
The sensitivity of fluorescence depends not only on the absorptivity of the sample, but on the intensity of the incident light and the quantum yield of the conversion of absorbed energy to fluorescent light. Under optimal conditions, fluorescent samples can be measured at a sensitivity of about 104 better than conventional absorbance spectroscopy. This sensitivity, however, is achieved at the cost of versatility. Few analytes fluoresce with the yield needed for wide application of the technique. Fluorescence spectroscopy is made practical for non-fluorescing analytes by tagging them with large (e.g., ca. 500 Dalton) fluorescent molecules, thus adding an additional costly step in the overall analysis and possibly altering the chemistry of the analyte in the process.
Although both conventional absorption spectroscopy and fluorescence spectroscopy rely on the absorption of light by the analyte, they differ significantly in their sensitivity and versatility. Absorption spectroscopy is easily applied to a wide variety of analytes, but has inherently poor sensitivity. Fluorescence spectroscopy is sensitive, but only for a limited number of molecules. An array-based analysis technique is needed that combines the strengths of these two spectroscopic approaches to yield both high sensitivity and wide applicability.
Photoacoustic spectroscopy (PAS) is based on the absorption of light energy by a molecule. The signal in PAS, however, is not detected by monitoring the transmittance or emission of light. Instead, in PAS, the signal is monitored by acoustic detection. Specifically, photoacoustic spectroscopy detection is based on the generation of acoustic waves as a consequence of light absorption. Absorption of light by a sample exposed thereto excites molecules in the sample to higher rotational/vibrational/electronic states. Return to the ground state releases the absorbed energy to the surrounding medium, either as light or heat. Collisions of the molecules transfer the rotational/vibrational energy to translational energy, i.e., heat. Modulation of the light intensity (turning the light on and off as the sample is exposed) causes the temperature of the sample to rise and fall periodically. The temperature variation of the sample is accompanied by a pressure variation that creates a sound wave (gas samples must be in a closed volume). The sound wave can be detected with a sensitive microphone.
Conventionally, a sample to be analyzed by photoacoustic spectroscopy is placed in a cuvette or other similar singular sample holder. Although obtaining the advantage of PAS analysis selectivity and sensitivity, the known singular sample analysis is slow and labor intensive. There is some experimental use of PAS to analyze multiple samples, as disclosed in U.S. application Ser. No. 10/061,235, now U.S. Pat. No. 6,870,626. There is also experimental use of PAS analysis utilizing conventional microtiter plates, as disclosed in U.S. application Ser. No. 10/002,624, now U.S. Pat. No. 6,873,415, which application is incorporated herein by reference. There is still a need, however, for development of sample array vessels desired specifically for PAS analysis and for analysis parameter methods desired for PAS analysis of such sample array vessels. Further, there is a need for sample array vessels including acoustic detectors operable with PAS systems and methods that allow for PAS sample analysis using the same.